In recent years, botulinum neurotoxins have become the standard agent in the treatment of focal dystonias and spastic indications. Treatment of patients generally involves injection of the neurotoxin into affected muscle tissue, bringing the agent near the neuromuscular end plate, i.e. close to the cellular receptor mediating its uptake into the nerve cell controlling said affected muscle. Various degrees of neurotoxin spread have been observed. This spread is thought to correlate with the injected amounts and the particular preparation of neurotoxin injected. Resulting from the spread, systematic side effects caused by the inhibition of acetylcholine release, may be observed at nearby muscle tissue. The incidents of unintended paralysis of untreated muscles can largely be avoided by reducing the injected doses to the therapeutically relevant level. Overdosing may also be a problem with regard to the patients' immune system, as the injected neurotoxin may trigger the formation of neutralizing antibodies. If this occurs, the neurotoxin will be inactivated without being able to relieve the involuntary muscle activity.
Differences in the dose equivalents or variations in the determined activity of preparations such as available sales products or batches produced during the manufacturing process, commonly a fermentation process, pose an increased risk for patients through possible side effects and the development of immunity. Therefore, it is of crucial importance to determine the biological activity of a clostridial neurotoxin contained in said sales products or production batches reliably (i.e. without significant variation) and as accurately as possible, in order to adjust the neurotoxin concentration to a reliable effective dose for the benefit of the patient. This may also serve as an incentive to the manufacturers to offer formulations allowing optimum exploitation of biological activity for different therapeutic purposes.
At present, the botulinum neurotoxin testing is predominantly performed using the mouse LD50 assay developed more than 40 year ago (see Boroff and Fleck, J. Bacteriol. 92 (1966) 1580-1581), which is accepted for potency testing by United States and European regulatory agencies. This assay involves dosing mice with dilutions of the sample of botulinum neurotoxin being tested and calculating the dilution at which 50% of the mice would be expected to die. Since this bioassay requires up to 100 mice for testing a single sample, and takes up to four days to generate results, there is a large need for alternative methods that are faster and more accurate, and/or reduce, cause less pain, distress, and/or replace use of animals such as mice. Any such alternative method, in order to be acceptable to regulatory agencies for the determination of potency, must be suitable for the intended purpose of the product in question and must be validated for sensitivity, specificity, reproducibility and robustness. For botulinum neurotoxin testing, a suitable potency assay must be used to determine the dose of the final product or to compare the relative activities of different lots. Because botulinum neurotoxin activity is dependent upon three functional domains within the protein molecule, an acceptable potency assay must account for the activity of all domains. For a discussion of issues relating to the unsolved problem of mouse LD50 assay replacement, see “Report on the ICCVAM-NICEATM/ECVAM Scientific Workshop on Alternative Methods to Refine, Reduce or Replace the Mouse LD50 Assay for Botulinum neurotoxin Testing”, NIH Publication Number 08-6416, February 2008.
For example, several cell-based assays using a Western Blot readout have been discussed in the context of identifying alternatives to the mouse LD50 assay. Eubanks et al. describe an antibody-based assay for detection of SNAP25 cleavage by BoNT/A in PC12 or Neuro-2A cells (Eubanks et al., FEBS Lett. 2005; 579: 5361-4). A similar assay is based on the detection of SNAP25197 in differentiated Neuro-2A cells (Fernandez-Salas et al., ABS-29 presented at: Basic and Therapeutic Aspects of Botulinum and Tetanus Toxins International Conference (Toxins 2005), Jun. 23-25, 2005, Denver, Colo.; Steward et al., ABS-76 presented at: Basic and Therapeutic Aspects of Botulinum and Tetanus Toxins International Conference (Toxins 2005), June 23-25, 2005, Denver, Colo.).
Alternatively, cellular assays were set up that monitor the neuronal network or neurotransmitter release on clostridial neurotoxin treatment. Chaddock et al. describe an assay based on the detection of potassium stimulated 3H-glycine release in a primary culture of fetal rat spinal cord cells on treatment with BoNT/A (Chaddock et al., Protein Expr Purif. 2002; 25: 219-28). Gross et al. used the reduction of spontaneous spiking and bursting as read-out in embryonic murine spinal cord and frontal cortex cells (Gross et al., Society for Neurosciences 2003, Abstract 122.9). That assay was originally developed by Keefer et al. using either BoNT/A or tetanus toxin (Keefer et al., Society for Neurosciences 2001, Abstract 1302).
Additionally, cellular assays were studied using Botulinum neurotoxin substrates carrying fluorescence markers. Dong et al. presented a FRET-assay in PC12 cells using SNAP25 or Synaptobrevin each labelled with two fluorescent proteins for monitoring BoNT/A and BoNT/B activity, respectively (Dong et al., Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 14701-6). A similar assay was set up by Steward et al. in differentiated Neuro-2A cells (Steward et al., International Conference on Basic and Therapeutic Aspects of Botulinum and Tetanus Toxins, 23-25 June 2005, Denver, USA: ABS-76).
The above referenced prior art methods, however, have not resulted in a method certified by regulatory authorities. Therefore, the out-of-time mouse killing assay must still be performed, and the need still exists to identify alternative assays.